Abstract Zika virus (ZIKV) is primarily a mosquito-transmitted virus but the concerns regarding sexual transmission has gained urgency as the alarming increase in the number of cases of microcephaly in ZIKV-infected women in the 2015-2016 global epidemic coincided with the detection of the virus in the semen. Recent reports of ZIKV in human seminal fluid for months after the clearance of viremia in as high as 56% of infected men together with low sperm count suggest the ability of ZIKV to enter into the immune privilege compartment of the testes and establish persistent infection. Lack of any specific therapeutic drugs to clear testicular infection and appropriate animal model to mimic human testicular ZIKV infection has brought an urgent need for the development of human in vitro systems to understand the associated mechanisms of persistence. Human testes immune environment is tightly governed by an elaborate communication network between different resident cell types including Sertoli cells (SCs) and Leydig cells (LCs) that produce key cytokines (TGF-? and TNF-?) and hormones (testosterone, inhibin and activin) required for the maintaining local immune homeostasis and supporting germ cells differentiation. Our recent study demonstrated that ZIKV infection of primary human SCs is more robust than Dengue virus (DENV), modulates pathways involved in innate immune response and SC-germ cells network thus suggesting that ZIKV may affect the function of seminiferous epithelium and antiviral response. We have recently developed a human 3D testicular organoid (HTO) culture system using cells present in the interstitial space (testosterone producing LCs) and seminiferous tubules (SCs, peritubular cells and spermatogonia stem cells) that supports differentiation of germ cells. Our preliminary studies demonstrate that ZIKV can productively infect these HTOs. This study will address the hypothesis that multiple human testicular cells support robust infection of ZIKV and that these HTOs can be used as a novel model system to assess the cell tropism of the virus and alterations in the key pathways uniquely associated with testicular immune response and germ cell survival. In the aim 1, we will determine replication kinetics of ZIKV, WNV and DENV and effect on overall HTO integrity, cell death and fate of spermatogonia stem cells. Aim 2 will determine the association of cell-type specific immune response pathways with levels of virus transcripts and testicular hormones in each partner cell. Our group is uniquely positioned to establish this multicellular 3D HTOs as a novel tool to delineate the mechanisms of ZIKV in testicular cells because of our expertise in the field of flavivirus immunity, organoid and germ cells culture. The data from this study will fill the fundamental gap of cell types that support long-term infection of ZIKV in the testes and open up opportunities to use HTOs for future mechanistic and translational studies including screening of anti-viral drugs to clear testes infection.